Dysfunctional serotonergic neuromodulation in mood-regulating circuits underlies many psychiatric diseases, thus understanding regulation of serotonin (5-HT) transmission is of fundamental importance. The 5-HT transporter (SERT) clears 5-HT from extracellular fluid with high-affinity, and is considered a primary controller of the strength and duration of 5-HT signaling. Our studies have revealed that organic cation transporter 3 (OCT3), a low-affinity, but high-capacity transporter of monoamines, plays a critical role in 5-HT clearance as well. Though circuits modulating arousal and emotion are highly complex, processing within the basolateral amygdala (BLA) is considered essential, especially for fear conditioning. The BLA receives dense input from 5-HT neurons in dorsal raphe nucleus (DRN), and BLA principal neurons have numerous fear-regulatory outputs, including dense projections to medial entorhinal cortex (mEC), which serves as a gateway for fear memory information flow into and out of hippocampus. Like SERT, OCT3 is highly expressed in BLA, ideally positioning these transporters to powerfully control extracellular 5-HT and its local neuro-modulatory efficacy. Proposed studies test the hypothesis that 5-HT clearance by OCT3 and SERT in BLA facilitates acquisition and consolidation of fear memory by buffering the rise of 5-HT that normally restrains BLA-mEC neuronal activation by excitatory fear memory-promoting limbic inputs. We posit that fear conditioning stimuli, which lead to fear memory, co-activate limbic and DRN 5-HT inputs to BLA along with activating the hypothalamic-pituitary-adrenal stress axis. OCT3 is potently inhibited by corticosterone, indicating that diminished OCT3 clearance allows 5-HT to rise high enough during fear conditioning to activate 5-HT receptors and effectively buffer limbic excitation of BLA-mEC neurons, decreasing their output and reducing fear memory. We will use state-of-the-art conditional gene deletion strategies to separately and collectively deplete SERT and OCT3 from DRN neurons, combined with optogenetic activation and inhibition of DRN 5-HT neurons projecting directly to BLA. AAV shRNA will be used to knockdown SERT and/or OCT3 on all cell types in BLA. These approaches will be used to determine the relative contributions of SERT and OCT3 to 1) 5-HT clearance in BLA in vivo using high-speed chronoamperometry; 2) 5-HT modulation of BLA-mEC neuronal activity using in vivo single neuron and whole-cell patch clamp recording in brain slices; 3) fear conditioning behavior. Because of their important roles in fear conditioning and 5-HT signaling in BLA, we will interrogate the functional contributions of 5-HT2A and 5-HT1A receptors in this circuit. Serotonergic neurotransmission potently modulates behavior, and its dysregulation is strongly implicated in psychiatric diseases. Proposed, discovery driven, studies will provide unprecedented mechanistic insights into the role 5-HT, and its regulation by SERT and OCT3, play specifically within the DRN-BLA-mEC fear conditioning hub.